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Alveolar macrophages (AM) in the lung have been documented to play pivotal roles in inflammation and fibrosis (silicosis) following inhalation of crystalline silica (CSiO2). In contrast, exposure to either titanium dioxide (TiO2) or amorphous silica (ASiO2) is considered relatively benign. The scavenger receptor macrophage receptor with collagenous structure (MARCO), expressed on AM, binds and internalizes environmental particles such as silica and TiO2. Only CSiO2 is toxic to AM, while ASiO2 and TiO2 are not. We hypothesize that differences in induction of pathology between toxic CSiO2 and nontoxic particles ASiO2 and TiO2 may be related to their differential binding to MARCO. In vitro studies with Chinese hamster ovary (CHO) cells transfected with human MARCO and mutants were conducted to better characterize MARCO-particulate (ASiO2, CSiO2, and TiO2) interactions. Results with MARCO-transfected CHO cells and MARCO-specific antibody demonstrated that the scavenger receptor cysteine-rich (SRCR) domain of MARCO was required for particle binding for all the tested particles. Only TiO2 required divalent cations (viz., Ca+2 and/or Mg+2) for binding to MARCO, and results from competitive binding studies supported the notion that TiO2 and both the silica particles bound to different motifs in SRCR domain of MARCO. The results also suggest that particle shape and/or crystal structure may be the determinants linking particle binding to MARCO and cytotoxicity. Taken together, these results demonstrate that the SRCR domain of MARCO is required for particle binding and that involvement of different regions of SRCR domain may distinguish downstream events following particle binding.
Prolonged occupational exposures (mining, construction, etc.) to inhaled crystalline silica (CSiO2) particles can lead to an irreversible and many times fatal fibrotic condition of the lungs called silicosis (Hamilton et al., 2008; Ng and Chan, 1992). Currently, no effective treatment exists for silicosis, which is a significant health problem throughout the world, particularly in developing nations (Saiyed and Tiwari, 2004).
Silica is one of the most abundant minerals found on the surface of earth; it exists in crystalline and amorphous forms: both of which contribute to occupational exposure. However, of the two silica types, CSiO2 is known to be the causative agent for silicosis. Although amorphous silica (ASiO2) has been reported to cause pulmonary inflammation following inhalation, it does not lead to silicosis (Merget et al., 2002; Reuzel et al., 1991). Similarly, another inorganic particle, titanium dioxide (TiO2), is relatively inert and is widely used in many industrial applications, as well as in medical and dental prosthesis (Driscoll et al., 1991; Lardot et al., 1998; Lindenschmidt et al., 1990).
Inhaled particles are initially encountered by the first line of defense, alveolar macrophages (AM) in the lungs. AM are cells that are primarily responsible for binding, ingestion, and ultimately clearance of inhaled particulate matter (Hamilton et al., 2008). When AM encounter CSiO2, they have been shown to rapidly engulf the particles and undergo apoptosis (Hamilton et al., 2008; Iyer et al., 1996). Consequently, it is possible that AM engulf both apoptotic bodies and free CSiO2 particles and then secrete proinflammatory cytokines or undergo apoptosis. This recurrent cycle of engulfment, apoptosis, and cytokine secretion can lead to recruitment of other inflammatory cells that contribute to prolonged inflammation and development of fibrosis (Beamer and Holian, 2007; McCabe, 2003). In contrast, ASiO2 and TiO2 (at sizes used in this study) do not induce macrophage apoptosis, suggesting that apoptosis could play a role in initiating the fibrotic development by silica (Arts et al., 2007; Iyer et al., 1996; Thibodeau et al., 2003). However, ultrafine (≤ 20 nm) TiO2 particles have been reported to induce release of fibrogenic mediators and induce apoptosis in fibroblasts (Rahman et al., 2002). This phenomenon is further supported by studies that indicate that the fibrogenic potential of a particle correlates with its ability to induce apoptosis in AM (Iyer et al., 1996; Rimal et al., 2005).
Scavenger receptors (SRs) are cell-surface glycoproteins capable of binding a broad spectrum of ligands including oxidized and acetylated lipoproteins and bacterial pathogens (Murphy et al., 2005). Recent studies have demonstrated that SR are involved in environmental particle binding and lung inflammation (Arredouani et al., 2006; Hamilton et al., 2006). One member of the SR family is macrophage receptor with collagenous structure (MARCO), which is mainly expressed by macrophages, dendritic cells, and certain endothelial cells. Studies have identified MARCO as a key receptor in recognizing CSiO2 and causing apoptosis in murine AM (Hamilton et al., 2006). Furthermore, MARCO has also been reported to be a receptor for TiO2 (Arredouani et al., 2005). However, even though MARCO binds both CSiO2 and TiO2, they show contrasting apoptotic and pathological outcomes (Thakur et al., 2008).
MARCO is a 210-kDa trimeric, type II membrane protein comprised of a short intracellular and a large extracellular domain, a transmembrane domain, and a C-terminal cysteine-rich (scavenger receptor cysteine-rich [SRCR]) domain (Elomaa et al., 1995; Kraal et al., 2000). The SRCR domain is an ancient, highly conserved domain consisting of 100 amino acid residues (Sarrias et al., 2004). Proteins expressing this highly conserved motif are classified into an SRCR superfamily and have a wide range of functions often associated with the innate immune system (Rast et al., 2006; Sarrias et al., 2004). The SRCR domain of MARCO has been shown to be a binding site for bacteria, lipopolysaccharide, and acetylated lipoproteins (Brannstrom et al., 2002; Chen et al., 2006). Recently, the SRCR domain of MARCO was crystallized and was found to possess a basic cluster containing several arginines (positively charged) and a separate acidic cluster containing a bound metal ion (negatively charged). Both clusters were reported to be involved in ligand binding (Ojala et al., 2007). Taking into consideration the negative surface charge of environmental particles such as silica and TiO2, it can be postulated that the positively charged arginines in the SRCR domain of MARCO might be important for binding of the particles. Although previous studies have focused on determining the role of MARCO in binding of environmental particles, the exact binding site has not been determined. Collectively, we hypothesize that the difference in induction of pathology between toxic CSiO2 and the nontoxic ASiO2 and TiO2 may be related to differential binding to MARCO and the signaling events triggered by these particle-MARCO interactions. The two primary goals of the present study were to identify the particle-binding domain of MARCO and to examine the parameters that influence particle binding to MARCO.
CD204-null mice on C57Bl/6 background were kindly provided by Dr Lester Kobzik (Harvard school of public Health, Boston, MA). Genotyping was carried out as described previously (Dahl et al., 2007). All mice were maintained in the University of Montana specific pathogen-free laboratory animal facility. The mice were maintained on an ovalbumin-free diet and given deionized water ad libitum. The University of Montana Institutional Animal Care and Use Committee approved all animal procedures.
CSiO2 (Min-U-Sil-5) obtained from Pennsylvania Sand Glass Corporation (Pittsburg, PA) was acid washed in 1M HCl at 100°C to remove metals and microbial contamination. The CSiO2 particles were then washed three times with sterile water and dried at 200°C to remove all water. TiO2 particles were purchased from Fischer Scientific (Pittsburg, PA) (T315-500). The 4′,6-diamidino-2-phenylindole (DAPI)−conjugated ASiO2 particles 1 μm in diameter were purchased from Postnova Analytics, Inc. (Salt Lake City, UT). The ASiO2 particles were washed three times with sterile phosphate-buffered solution (PBS) to remove the shipping medium. For all binding and cytotoxicity experiments, the particles were suspended in either PBS-azide buffer (PAB) (0.1% sodium azide, 1% fetal bovine serum [FBS] in PBS) or Ham F-12 media at 2.5 mg/ml. The stock suspensions were dispersed by sonic disruption for 1 min before each experiment. For all experiments, CSiO2 and ASiO2 were used at a concentration of 150 μg/ml. Since TiO2 particles were smaller in size (100–200 nm) as compared to CSiO2 and ASiO2 (1–2.5 μm), the cells were treated with 25 or 75 μg/ml in an effort to keep the number as well as the surface area of particles comparable, but sufficient for binding studies.
Mice were euthanized by a lethal injection of Euthasol. The lungs were removed with the heart and then lavaged with five 1-ml aliquots of cold PBS. Pooled cells were centrifuged at 400 μg for 5 min. The lavage fluid was aspirated and discarded. The cell pellet was resuspended in 1 ml of RPMI 1640 culture media supplemented with 10% fetal bovine serum, 100 IU penicillin, 100 μg/ml streptomycin (Mediatech, Inc., Herdon, VA). Total lavage cells were enumerated using a Z1 Coulter Particle Counter (Beckman Coulter, Fullerton, CA). The cells were adjusted to 106 per milliliter and added to 0.65-ml sterile polypropylene tubes at 500 μl/tube as described previously (Scheule et al., 1992). Particulates were added, and the cells were cultured in a tumbling suspension culture for 4 h at 37°C in a water-jacketed CO2 (5%) incubator (ThermoForma, Mariette, OH).
Isolated AM were cultured in suspension (106 cells/ml) with different concentrations of particles for 4 h at 37°C. At the end of this period, 10 μl of the culture supernatant was removed and mixed with 10 μl of 0.4% trypan blue solution (Sigma, St. Louis, MO). The resulting mixture was added to a hemocytometer, and the cells were examined by light microscopy. One hundred random cells were counted per sample, and cells that appeared to contain blue dye were considered dead. Data were expressed as percent of cells excluding trypan blue dye (percent living cells).
Chinese hamster ovary (CHO) cells (American Type Culture Collection, Manassas, VA) were cultured in HAM F-12 medium with 2mM L-glutamine (Mediatech, Inc.) containing 10% heat-inactivated FBS, 100 IU penicillin, 100 μg/ml streptomycin (Mediatech, Inc.). CHO cells were transiently transfected with pcDNA 3.1 (E), full-length human MARCO (M), truncated MARCO (Mt) lacking the entire SRCR domain, MARCO with only the initial 22 amino acids in the SRCR domain (M442), or MARCO mutant with only 11 amino acids (lacking the “RGR” motif) in the SRCR domain (M431). Scavenger receptor A I (SRA I) was included as an expression control. All transfections were conducted using Lipofectamine 2000 as per manufacturer's instructions (Invitrogen, Carlsbad, CA). Transfection efficiency of the full-length MARCO and the various mutants was determined to be 30–40% by staining the cells for MARCO expression using human MARCO-specific antibody (PLK-1) kindly provided by Dr Lester Kobzik (Harvard School of Public Health, Boston, MA) and isotype control (IgG3) plus fluorescein isothiocyanate−conjugated secondary antibody (Southern Biotechnology Associates, Birmingham, AL). Analysis was done using FACSAria flow cytometer using Diva software (version 4.1.2; BD Biosciences, San Jose, CA). All the experiments were conducted 36–40 h following transient transfections.
Transiently transfected CHO cells were harvested using trypsin, and the cells were resuspended in 1 ml of PAB and counted. Cells, 1 × 106, were treated with or without 10 μg/ml of monoclonal antibody against the SRCR domain of human MARCO (PLK-1) or 10 μg/ml isotype control IgG3 on ice for 15 min. The cells were then treated with different concentrations of CSiO2, TiO2, and ASiO2 for 30 min at 37°C in tumbling suspension culture. Particle binding was then measured as an increase in mean side scatter (nonfluorescent CSiO2 and TiO2) as previously described (Hamilton et al., 2006; Palecanda and Kobzik, 2000) and as increase in DAPI-positive cells (fluorescent DAPI ASiO2 particles) by FACSAria flow cytometer using Diva software (version 4.1.2; BD Biosciences). The analysis of the CSiO2 and TiO2 particle binding included changes in side scatter of all cells (transfected and untransfected).
Transiently transfected CHO cells (grown in 10-cm2 culture dishes) were washed twice with ice-cold PBS containing 1mM CaCl2 and 1mM MgCl2 (Ca+2/Mg+2) and then incubated on ice with 0.5 mg/ml Biotin (Pierce, Rockford, IL) for 15 min. Biotin treatment was repeated again for 15 min on ice. The cells were then washed once with ice-cold PBS (Ca+2/Mg+2) and lysed in cell lysis buffer. The cell lysate was then centrifuged and transferred to 1.5-ml eppendorf tubes. Forty microliters of neutravidin beads (Pierce) were added to the cleared lysate and rotated at 4°C for 2 h. The beads were then washed four times with the ice-cold cell lysis buffer. The protein was eluted from the beads with 2× sample buffer and denatured by heating at 70°C for 10 min. The 30 μl of bead-free lysate was then fractionated by 10% Bis-Tris NuPAGE gel (Invitrogen) and transferred to nitrocellulose membrane. The nitrocellulose membrane was incubated with monoclonal antibody against the intracellular domain of human MARCO. The membrane was incubated with an anti-rabbit horseradish peroxidase−labeled secondary antibody (R&D Systems, Minneapolis, MN), and protein signals were visualized with a chemiluminescent reagent (ECL Plus Western Blotting detection system; Amersham Biosciences, Piscataway, NJ).
Transiently transfected CHO cells were harvested using trypsin, and the cells were resuspended in Ham F-12 media and counted. Cells, 1 × 106, were treated with CSiO2, ASiO2, or TiO2 for 15 min in a tumbling suspension culture at 37°C to ensure particle binding to the cells. The treated cells were then plated in six-well plates at 37°C or 6 h. After 6 h, the cells were trypsinized, centrifuged, and resuspended in 1 ml of PBS, followed by addition of 1 μm of propidium iodide (PI) and 1 μm YOPRO-1, a DNA intercalant dye (Idziorek et al., 1995) that stains only apoptotic cells (Molecular Probes, Eugene, OR) for 20 min. The percentages of apoptotic (Yopro-1 positive) and late apoptotic (Yopro-1 and PI positive) cells were immediately determined by FACSAria flow cytometer using FACS Diva software (version 4.1.2; BD Biosciences).
Transiently transfected CHO cells were harvested using trypsin, and the cells were resuspended in 1 ml of PAB and counted. Cells, 1 × 106, were pretreated with 50 μg/ml of CSiO2 or 75 μg/ml of TiO2 for 15 min prior to different concentrations of ASiO2 exposure for 30 min at 37°C in tumbling suspension culture. The uptake of the ASiO2 particles was immediately measured as an increase in DAPI-fluorescent cells by FACSAria flow cytometer using Diva software (version 4.1.2; BD Biosciences).
Transiently transfected CHO cells were harvested using trypsin, and the cells were resuspended in 5 ml of Dulbecco PBS (dPBS) (Ca+2 and Mg+2 free) (Invitrogen) supplemented with 2mM ethylenediaminetetraacetic acid (EDTA) to chelate any remaining divalent cations present in the cell suspension. The cells were then washed twice with 5 ml of dPBS to remove residual EDTA. Cells, 106 per ml, were suspended in dPBS and treated with TiO2 (25 or 75 μg/ml), CSiO2 (150 μg/ml), and ASiO2 (150 μg/ml) in presence or absence of 5mM CaCl2 and/or 5mM MgCl2 (final concentration). The effect of divalent cations (Ca+2 and Mg+2) on particle binding was then measured as an increase in mean side scatter (SSc) by FACSAria flow cytometer using Diva software (version 4.1.2; BD Biosciences).
Particle size was measured by scanning electron microscopy (SEM), and to study the effect of the suspension media, the size was analyzed using light scattering techniques by Dr Nianqiang Wu (University of West Virginia, Morgantown, WV). Hitachi S4700 SEM was used to image the particles. Prior to SEM observation, the particle suspension was dropped on the surface of an Si wafer and then dried in ambient condition. The size of CSiO2 particles was measured by Malvern Mastersizer 2000 particle size analyzer. The particles were suspended in deionized water, PAB, or Ham F-12 media, sonicated, and then analyzed using Nanosizer and Microsizer instruments at rool temperature. The surface charge on the particles was measured by calculating the zeta potential of the particle (samples were analyzed by Colloidal Sciences Laboratory, Westampton, NJ).
Data were analyzed using the Prism Software, version 4 (GraphPad Prism, San Diego, CA). The significance of differences between treatment groups and controls was determined using one-way ANOVA or two-way ANOVA in conjunction with Bonferroni post hoc analysis depending on the experiment. Data are represented as mean ± SE. A value of p < 0.05 was considered significant.
To confirm previous reports of relative toxicity of CSiO2, ASiO2 and TiO2, the effect of these particles on AM viability was analyzed. Primary AM from CD204-/- mice were treated with different concentrations ASiO2, CSiO2 and TiO2 for 4 h in suspension-culture and changes in cell viability were measured using trypan blue exclusion assay (Hamilton et al., 2006; Iyer et al., 1996b). As expected, treatment with CSiO2 caused significant loss in cell viability while treatment with TiO2 and ASiO2 were relatively nontoxic (Fig. 1).
The SR MARCO is an important player in binding of both toxic CSiO2 and nontoxic TiO2 (Hamilton et al., 2006; Palecanda et al., 1999). Therefore, to begin understanding the role of MARCO in the contrasting effects of these particles, their binding patterns to MARCO were analyzed (Fig. 2). Initial studies were performed using CHO cells transiently transfected with full-length human MARCO (M) or empty vector, pretreated with or without MARCO antibody (PLK-1) or isotype control IgG3, followed by treatment with different concentrations of the three particles. Binding experiments were performed using a flow cytometric assay (Palecanda and Kobzik, 2000), wherein an increase in mean side scatter intensity was used as a marker for binding of CSiO2 and TiO2. For the DAPI-conjugated ASiO2 particles, increase in percentages of DAPI-positive cells was used as a measure of binding. The results showed that all three inorganic particles bound to MARCO (Fig. 2). Further, to define the specificity of particle bindings to MARCO, binding studies were conducted following pretreatment with MARCO-specific blocking antibody (Fig. 2). In each case, the MARCO antibody efficiently blocked binding for all three inorganic particles while the isotype control had no effect (Fig. 2; data not shown), suggesting that the SRCR domain of MARCO contained the particle-binding sites.
In order to confirm the requirement of the SRCR domain of MARCO in binding of particles, CHO cells were transiently transfected with full-length or truncated forms of MARCO. The truncated mutants either lacked the entire SRCR domain of MARCO (Mt) or only contained the first 22 amino acid residues of the SRCR domain (M442) that was reported to contain the bacteria binding RGR motif (Brannstrom et al., 2002). Cell-surface expression of the truncated mutants was analyzed by surface biotinylation (Fig. 3). CHO cells were transfected with full-length and truncated mutants, followed by precipitation of biotinylated proteins on ice and Western blotting as described in the “Materials and Methods” section. The nitrocellulose membranes were then probed with an antibody against the intracellular domain of MARCO. The results indicated that the mutants M442 and Mt were expressed on the surface (lanes 2 and 3) at higher levels compared to the full-length MARCO (lane 4). The increased expression of the mutants is similar to previously published results (Elomaa et al., 1998). As controls, SRA I- and mock-transfected cells were probed with the same antibody. As expected, streptavidin-agarose was not found to precipitate any biotinylated MARCO for mock- (lane 5) or SRA I (lane 1)-transfected cells.
The role of the SRCR domain of MARCO in particle binding was studied using transfected cells incubated with the three inorganic particles measured using flow cytometry as described above. The results showed that cells expressing the mutant lacking the SRCR domain of MARCO (Mt) did not bind any of the particles, indicating that the SRCR domain of MARCO is a common required binding domain for all the tested particles. The truncated protein (M442) with the RGR motif was not sufficient to bind CSiO2 and TiO2 (Fig. 4A). However, the ASiO2 particles bound to M442-expressing cells although the binding was significantly less than the full-length MARCO-expressing cells (Fig. 4B). Another mutant containing the first 11 amino acids of SRCR domain and lacking the RGR motif (M431) did not bind the ASiO2 particles (data not shown). These results showed that the RGR motif (found capable for supporting bacterial binding) in the SRCR domain of MARCO is not sufficient for binding of CSiO2 and TiO2, but the motif appears to play a partial role in ASiO2 binding.
CSiO2 has been reported to induce both apoptosis and necrosis in murine and human AM (Iyer and Holian, 1997; Iyer et al., 1996). In contrast, TiO2 and ASiO2 have been reported to be relatively benign (see Fig. 1). All three inorganic particles require the SRCR domain of MARCO for binding (see Figs. 4A and 4B). To establish the functional contribution of the SRCR domain of MARCO to cytotoxicity in the transfected CHO cell model, the full-length MARCO- and MARCO-variant−transfected cells were treated with CSiO2 or TiO2 for 6 h. The cells were stained with 1 μm of YOPRO-1 and PI, and the percentages of viable and apoptotic cells were analyzed by flow cytometry. As anticipated, CSiO2 induced significant apoptosis in full-length MARCO-transfected cells compared to the mock- and Mt-transfected cells (Fig. 5A). In contrast, TiO2 particles did not induce apoptosis with any of the transfectants (data not shown). The results support the role of the SRCR domain of MARCO in binding and apoptosis by CSiO2.
Competitive binding studies were conducted to determine if all the tested particles had overlapping binding domains or whether they use different binding motifs in the same SRCR domain. The transfected cells were pretreated with CSiO2 or TiO2, 15 min prior to incubation with different concentrations of ASiO2 particles. CSiO2 significantly inhibited ASiO2 binding (Fig. 6A). The TiO2 particles, however, did not inhibit the binding of ASiO2 particles to MARCO (Fig. 6B). The results strengthened the hypothesis that TiO2 and CSiO2 bind to different regions on the SRCR domain of MARCO. Since all particles increase the side scatter of cells when they bind, the reverse experiment could not be conducted, namely if ASiO2 effectively inhibits CSiO2 binding.
The crystalline structure of SRCR domain of murine MARCO was recently found to contain Mg+2 in its acidic amino acid cluster (Ojala et al., 2007). Also, the presence of divalent cations was found to be necessary for binding of some MARCO ligands (Ojala et al., 2007). To date, no studies have been conducted examining the role of divalent cations in particle binding to MARCO. Therefore, the binding of the three particles to MARCO-transfected CHO cells was measured in the presence or absence of exogenous cations (5mM of CaCl2, MgCl2, or both). The binding of crystalline and amorphous forms of silica was not affected by the presence or absence of divalent cations (Figs. 7B−C). In contrast, no significant TiO2 binding was observed in the absence of the divalent cations or in the presence of EDTA (added to ensure complete divalent cation removal, see the “Materials and Methods” section) (Fig. 7A). The addition of CaCl2 and/or MgCl2 allowed the binding of TiO2 to MARCO (Fig. 7A). These findings indicate that the TiO2 interactions with MARCO specifically require divalent cations, while CSiO2 and ASiO2 do not.
In order to better assess the role of physical properties of the particles on their binding, the influence of properties such as particle size, shape, and zeta potential (measure of the surface charge) on interaction with MARCO was examined (Table 1). SEM and light scattering analysis showed that the overall diameter of all the inorganic particles ranged from 200 nm to 2.5 μm. The TiO2 particles were the smallest and had a uniform diameter of 100–200 nm. ASiO2 particles were 1 μm in size, according to the specifications of vendor (Postnova Analytics). The CSiO2 was found to be more heterogeneous in composition with both relatively large and small particles. The large particles were about 1–2.5 μm in size, while the smaller particles ranged from 200 nm to 1.0 μm. Suspending medium (media, H2O, PAB) had no significant effect on the particle size (Table 1).
Analysis of the SEM data also revealed the shape of the particles. The TiO2 particles were found to be spherical, while CSiO2 particles were found to be irregular in shape (Table 1). The ASiO2 particles, according to the vendor specification (and visual microscopic examination), were spherical in shape. The results obtained from the zeta potential measurement of the particles (Table 1) showed that the TiO2 particles were most negative with zeta potential (−) 47.9 mV, whereas CSiO2 and ASiO2 had a similar zeta potential, (−) 16.2 and (−) 17.8 mV, respectively.
Recent studies provided evidence that in AM from C57Bl/6 mice, MARCO plays a predominant role in binding of toxic CSiO2 particles (Hamilton et al., 2006). The ability of MARCO to bind inert nontoxic TiO2 particles was first reported by Kobzik and coworkers (Palecanda et al., 1999). These observations raise an important question as to why, despite binding to a common receptor MARCO, certain inorganic particles such as CSiO2 are toxic to the AM while TiO2 particles are not (Fig. 1). We hypothesized that the differences in the apoptotic outcome in response to these inorganic particles may, at least in part, be related to differences in binding of these particles to MARCO. To understand the differences in binding of environmental particles to MARCO, the purpose of this study was to define the particle-binding domain of MARCO and map some of the determinants for individual particle binding to MARCO.
To explore the possibility that crystalline and amorphous forms of silica, as well as TiO2, bind to distinct motifs in the receptor MARCO, a transfected cell line model was developed. For these studies, CHO cells expressing full-length MARCO or various MARCO mutants were used. The cell-surface expression of the full-length MARCO and mutants was confirmed by cell-surface biotinylation (Fig. 3). The results of the binding studies conducted with full-length MARCO-transfected cells showed that all three particles bound to MARCO (Fig. 2). The fact that the MARCO-specific antibody, which binds to an epitope in the SRCR domain, significantly inhibited the binding of all three particles to MARCO suggested that the SRCR domain was the particle-binding domain of MARCO. This finding was confirmed by the observation that the MARCO mutant without the SRCR domain failed to bind any of the particles (Figs. 4A and 4B). Consequently, the data established that all these particles require the SRCR domain for binding. Apoptosis assays with transfected CHO cells further showed that the CSiO2 binding to the SRCR domain of MARCO was required for its cytotoxicity (Fig. 5), whereas treatment with ASiO2 and TiO2 did not induce apoptosis in the full-length MARCO-transfected cells (data not shown) despite efficient binding (Fig. 2). The results support the hypothesis that the SRCR domain is the binding domain for environmental particles.
The next question was whether the RGR motif within the SRCR domain would be sufficient for particle binding. The M442 mutant containing the RGR motif showed significant ability (distinctly reduced compared to full-length MARCO) to bind only the ASiO2 particles (Fig. 4B). The CSiO2 and TiO2 particles did not bind to the M442 mutant−expressing cells. The RGR motif within the SRCR domain has been previously shown to be sufficient for bacterial binding (Brannstrom et al., 2002). Importantly, this finding suggests that ASiO2 is unique with respect to the RGR motif in contrast to CSiO2 and TiO2 and, hence, binds distinctly to MARCO.
Competitive binding studies were conducted to further investigate how each particle binds to the SRCR domain of MARCO. For these studies, cells were pretreated with CSiO2 or TiO2 particles prior to incubation with ASiO2 particles. Considering relatively large sizes of the particles, the complete inhibition of ASiO2 binding by CSiO2 was not unexpected (Fig. 6A). Furthermore, this observation does not negate the proposed role of the RGR motif as being sufficient for ASiO2 binding. It should be kept in mind while interpreting these results that all these particles are very large with respect to MARCO. Therefore, the observation that TiO2 did not completely block ASiO2 binding is more difficult to explain (Fig. 6B). Nevertheless, it indicates a divergence in the requirements between both the silica particles and TiO2 in binding to MARCO. This is not a classic case of ligand receptor binding but may require multiple MARCO receptors interacting with these rather large ligands.
Divalent cation-binding properties of certain receptors such as low density lipoprotein receptors are often exploited in nature to regulate complex biological events such as receptor-ligand interaction, endocytosis, and dissociation of the ligand from the receptor (Dirlam-Schatz and Attie, 1998). Recently, the SRCR domain of MARCO was shown to contain an acidic and a distinct basic cluster of amino acids, both the clusters were reported to be important for ligand binding. The crystallized SRCR domain of MARCO contained a divalent cation in the acidic cluster (Ojala et al., 2007). In the current study, divalent cations (Ca+2, Mg+2) were necessary only for TiO2 binding (Fig. 7A), whereas the other two inorganic particles (CSiO2 or ASiO2) did not depend on the presence of divalent cations (Figs. 7B−D). Calcium binding to the cysteine-rich domain of a particular protein has been shown to stabilize the protein conformation (Handford et al., 1990; Knott et al., 1996; Thielens et al., 1988). Bound calcium might cause a conformational change in the SRCR domain and expose certain amino acid residues leading to more efficient binding. The data suggest that either the TiO2 binds to the acidic cluster (containing the divalent cations) of the SRCR domain or the divalent cation binding to the SRCR domain leads to a distinct conformational changes in the binding domain facilitating TiO2 binding. It should be noted that changes in dispersion medium such as divalent cations will affect the zeta potential of all three particles. However, addition of divalent cations would affect all particles in a relatively similar manner. Therefore, it is most likely that the divalent cations in this study act on the SRCR domain of MARCO. Taken together, the results (Figs. 4, ,6,6, and and7)7) emphasize that the three different particles in the study show significant differences in binding to MARCO.
The differences in the pathological outcomes after exposure to each particle are speculated to be related to the differences in the physical properties of the particles. (Johnston et al., 2000; Thakur et al., 2008). Therefore, the particles were characterized and analyzed for their size, shape, and surface charge (Table 1). The analysis suggested that there were no major differences in size of the particles. The overall surface charge or the zeta potential of the silica particles was essentially identical (−) 16.2 to (−) 17.8 mV. The TiO2 particles were the most negative particles with zeta potential (−) 47.9. While the net surface charge of the particles could be measured by zeta potential measurements, the surface charge distribution (order) could not be determined. While it appears that TiO2 differs from the two silica particles in how it binds to MARCO and, therefore, could explain the difference in toxicity between TiO2 and CSiO2, the difference between ASiO2 and CSiO2 appears to be subtle. They are similar in relative size and surface charge but differ in shape and crystal structure (Table 1). It is possible that one or both the properties contribute to the difference in toxicity between the two silica particles. The only difference in binding appeared to be the sufficiency of the RGR motif for ASiO2 binding. The assumption made in these studies is that the two silica particles cause difference in conformational change in MARCO such that CSiO2 causes apoptosis while ASiO2 does not. As stated above, the difference in shape and crystal structure could affect silica binding to MARCO causing differences in conformation and downstream signaling. Without more data, the importance of shape or crystal structure is speculative. As yet the signaling pathways initiated by receptor MARCO are not completely elucidated, which makes the aforementioned theory difficult to test experimentally.
We propose that factors such as presence of divalent cations, shape of the particle, and crystal structure (the distribution or the order of surface charge) on the particle may play an important role in determining toxic or nontoxic binding of particles. Studies conducted with more diverse sizes and surface charge, of one particle could further strengthen these conclusions. Nevertheless, the results obtained in the current study provide strong support to the notion that environmental particles bind to distinct motifs in the SRCR domain of MARCO, which is influenced by individual physical properties. The implications of these observations are that each particle-MARCO interaction may trigger unique conformational changes in the receptor, which might influence the recruitment of different intracellular proteins leading to diverse biological responses.
The National Center for Research Resources [P20 RR017670]; the National Institute of Environmental Health Sciences [R01 ES015294].
The authors thank Nianqiang “Nick” Wu (University of West Virginia) for measuring the sizes of CSiO2 and TiO2 using SEM and light scattering techniques.The authors would also like to thank Sudhakar Agnihothram, Division of Biological Sciences, University of Montana for his technical expertise in molecular biology.